Nonlinear relation between concentration and fluorescence emission of protoporphyrin IX in calibrated phantoms

Abstract

5-ALA-induced protoporphyrin IX (PpIX) has shown its relevance in medical assisting techniques, notably in the detection of glioma (brain tumors). Validation of instruments on phantoms is mandatory and a standardization procedure has recently been proposed. This procedure yields phantoms recipes to realize a linear relationship between PpIX concentration and fluorescence emission intensity. The present study puts forward phantoms where this linear relationship cannot be used. We propose a model that considers two states of PpIX, corresponding to two different aggregates of PpIX, with fluorescence spectra peaking at 634 and 620 nm, respectively. We characterize the influence of these two states on PpIX fluorescence emission spectra in phantoms with steady concentration of PpIX and various microenvironment parameters (surfactant, Intralipid or bovine blood concentration, and pH). We show that, with fixed PpIX concentration, a modification of the microenvironment induces a variation of the emitted spectrum, notably a shift in its central wavelength. We show that this modification reveals a variation of proportions of the two states. This establishes phantom microenvironment regimes where the usual single state model is biased while a linear combination of the two spectra enables accurate recovering of any measured spectra.

Spectral complexity of 5-ALA induced PpIX fluorescence in guided surgery: a clinical study towards the discrimination of healthy tissue and margin boundaries in high and low grade gliomas

“…The presence of a second peak of fluorescence at 620 nm, which produces this shift, has been shown in tissues [32,[39][40][41] or in cell culture [42,43]. It is known that such a shift can be induced by a change in PpIX aggregation, which modifies the proportions in State 634 and State 620 of PpIX [35][36][37][38]. Hence, the blue shift measured in this study in low-density cell tumor region is another indication that HGG margins and LGG could produce a different aggregation of PpIX, due to a microenvironment favoring State 620 of PpIX.…”

“…It should be pointed that the fluorescence quantum yield of the two aggregates of PpIX are very different, the one of State 634 being far higher than the one of State 620 [36]. Then, a decrease in fluorescence intensity might not only reveal a decrease in PpIX concentration but also a change of PpIX aggregate, in favor of State 620 [37]. Our results suggest that the decrease of PpIX fluorescence intensity in low-density tumor cells regions [21][22][23] attributed mainly to low PpIX concentrations could also be linked to a different aggregation of PpIX in these regions, due to a microenvironment favoring the PpIX State 620.…”

“…The inner close to curre always at the probe, excitin scrimination of GG. [37]. We u by [44] cleaved so that excited tissue area and emitting tissue area are the same.…”

“…The presence of the second peak of fluorescence of PpIX at 620 nm is known in solution and closely linked with the chemical microenvironment [35][36][37][38]. In particular, PpIX is known to be pH related [32].…”

“…The fluorescence emission model used in the parametric data analysis has been discussed previously in detail [32,37]. However further improvements are added in these study with the fitting of lipofuscin.…”